Gene optimized hantaan virus M segment DNA vaccine for hemorrhagic fever with renal syndrome

ABSTRACT

A synthetic, codon-optimized Hantaan virus (HTNV) full-length M gene open reading frame that consists of a unique nucleotide sequence encoding HTNV proteins. This synthetic gene was cloned into a plasmid to form the first optimized HTNV full-length M gene that elicits neutralizing antibodies in animals when delivered in combination with a similarly optimized Puumala virus (PUUV) DNA vaccine. The invention obviates the need for an extraneous gene sequence that was previously required for expression of the non-optimized HTNV gene. The synthetic gene is engineered into a molecular vaccine system to prevent hemorrhagic fever with renal syndrome (HFRS) caused by infection with HTNV, SEOV, or DOBV. Alternatively, it can be combined with the optimized PUUV DNA vaccine to protect against HFRS caused by any hantavirus.

This application claims priority and is a divisional application of U.S.Ser. No. 14/756,608 filed Sep. 24, 2015 which is a continuation ofPCT/2013/000098 filed Mar. 28, 2013.

GOVERNMENT INTEREST

The invention described herein may be manufactured, used and licensed byor for the U.S. Government.

BACKGROUND OF THE INVENTION

The invention relates to a vaccine for hemorrhagic fever with renalsyndrome caused by hantavirus infections.

The Hantavirus genus of the family Bunyaviridae includes a number ofrodent-borne viruses that can cause hemorrhagic fever with renalsyndrome (HFRS) or hantavirus pulmonary syndrome (HPS). At least fourhantaviruses cause HFRS: Hantaan (HTNV), Seoul (SEOV), Dobrava (DOBV),and Puumala (PUUV) viruses. HFRS presents with sudden fever, chills,nausea, headache, and backache. Early symptoms of severe HFRS often alsoinclude facial flushing, conjunctivitis, and petechial rash. Death canoccur due to vascular leakage leading to low blood pressure, acuteshock, and renal failure. There are no FDA-licensed vaccines for HFRS,but an inactivated, rodent-brain-derived HTNV vaccine is commerciallyavailable in Korea, and several inactivated cell culture-derived HTNVand SEOV vaccines have been developed in China [1,2].

Despite the use of these vaccines for more than a decade, HFRS remains asignificant public health threat in Asia with thousands of hospitalizedcases reported each year in China [3-5]. Several hundred to thousands ofHFRS cases due to PUUV or DOBV infections are reported each year inEurope, Scandinavia, and Russia, with the greatest incidences observedin Finland (˜25,000 cases from 1979 to 2006) and western Russia (˜89,000cases from 1996 [6]. Inactivated vaccines have not been developed inEurope, in part because PUUV is difficult to grow in cell culture tohigh enough titers for scale-up, and rodent brain-derived vaccines arenot considered desirable. Moreover, because DOBV and PUUV both causeHFRS in the same geographic region, and because there is little or nocross-protective immunity between PUUV and the other HFRS-causinghantaviruses [7,8], a comprehensive vaccine for European HFRS will needto elicit protective immunity to both viruses.

To date, two recombinant DNA vaccines for HFRS have been tested in earlyclinical studies. The first tested was a vaccinia virus (VACV)-vectoredvaccine, developed and evaluated in Phases 1 and 2 clinical studies byUSAMRIID [9,10]. The vaccine expressed two of the three gene segments ofHTNV: the M segment, which encodes the envelope glycoproteins (Gn andGc), and the S segment, which encodes the nucleocapsid protein (N). Ingeneral, animal studies have shown that neutralizing antibodies to Gnand Gc are the best measurable correlate of protective immunity [8,11-13]. This earlier study found that the recombinant VACV vaccineelicited neutralizing antibodies against HTNV in VACV-naïve individuals,but was poorly immunogenic in VACV-immune volunteers [9]. Consequently,the vaccine developers changed strategies to a DNA vaccine platform,which was not adversely affected by preexisting vector immunity andwhich offered additional flexibility for producing combination vaccines.In addition to flexibility, DNA is an attractive vaccine platform interms of ease of engineering and manufacturing as well as safety.

USAMRIID investigators have so far conducted two Phase 1 clinicalstudies with DNA vaccines for HFRS using DNA derived from HTNV and fromPUUV M segments. The two-part DNA vaccine strategy was used becausevaccination with the HTNV M gene-based DNA vaccine protects animals frominfection with HTNV, DOBV and SEOV, but not from PUUV infection. PUUV Mgene-based DNA vaccine protects against infection with PUUV [7-8].

The first two clinical studies of the HTNV and PUUV DNA vaccines wereperformed using a PUUV M segment vaccine that was genetically optimized(US 2010/0323024A1, incorporated herein by reference in its entirety).The HTNV component, however, was not optimized, because unlike the PUUVDNA, which required optimization for gene expression, the HTNV DNAconstruct showed strong gene expression without optimization. It couldnot be anticipated, therefore, that a similar optimization was eithernecessary or would offer a benefit over the non-optimized DNA forimmunogenicity. Further and formerly, an extraneous gene sequence wasrequired for the expression of the non-optimized HTNV gene, U.S. Pat.No. 7,217,812, incorporated by reference, herein, in its entirety.

In the first clinical study of the DNA vaccines, HTNV and PUUV Msegments were delivered by particle mediated epidermal delivery (PMED).The advantage of intraepidermal delivery of the vaccine is twofold. TheDNA is easily taken up by cells at the site of delivery or by cells inthe draining lymph nodes where the antigen encoded by those cells isreprocessed by specialized antigen-presenting cells to elicit an immuneresponse, and this approach uses 1000-fold less DNA than needleadministration.

The vaccines were given as separate administrations because of resultsfrom animal studies, which showed that if the HTNV vaccine is mixed withthe PUUV vaccine, then only neutralizing antibodies to PUUV are elicited[25]. This finding was not expected, because it was possible to obtainstrong responses to the individual vaccines or to both vaccines whenthey were delivered simultaneously, but as separate inoculations, to asingle animal. In addition, it was not possible to overcome thisinterference by adjusting the ratio of HTNV: PUUV DNA even as high as10:1 (FIG. 8B). Other attempts to produce modified constructs that werechimeras of both the HTNV and PUUV genes also failed to elicit antibodyresponses to both HTNV and PUUV (unpublished information). The outcomeof the interference study is summarized in Example 1.

In a second Phase 1 clinical study of the same two DNA vaccines, theDNAs were given separately or as a mixture by intramuscularelectroporation (IM-EP). With this delivery method, the vaccines areinjected into muscles and a rapid electrical pulse is applied tofacilitate uptake of the DNA into the muscle cells. Because a largernumber of host cells receive the vaccines than when they are deliveredby PMED, it was anticipated that there might be some response to bothvaccines. As expected, however, interference was still a problem inindividuals receiving the mixed vaccines, with better responses obtainedto the PUUV vaccine than to the HTNV vaccine as shown in Example 2.

Delivery of the vaccine can also be by nanoparticle encapsulation of thevaccine via various methods, including aerosol delivery of thenanoparticles.

The present invention provides a combination vaccine to protect againstHFRS. The invention consists of an optimized HTNV M segment vaccine,which solves the problem of interference in the bivalent vaccine. Unlikethe non-optimized HTNV vaccine used in previous studies, the vaccine ofthe invention can be mixed with a similarly optimized PUUV-based vaccineto elicit neutralizing antibodies against both viruses. The inventionprovides a safe, economical, flexible and effective vaccine for theprotection of humans from HFRS caused by infection with HTNV, SEOV, PUUVand/or DOBV.

SUMMARY OF THE INVENTION

The invention is a synthetic, optimized HTNV M segment DNA vaccine thatis superior to the earlier non-optimized HTNV DNA vaccine and can beused by itself to prevent HFRS caused by three hantaviruses: (HTNV, SEOVor DOBV) or in combination with the optimized PUUV DNA vaccine toprotect from all four HFRS causing hantaviruses. The synthetic optimizedHTNV DNA does not require extraneous, superfluous nucleotides forexpression and immunogenicity and can be delivered as a mixture withother hantavirus vaccines without reduced immunogenicity or protectiveefficacy in animal models. To improve the vaccine component, the HTNVDNA vaccine was optimized to maximize mammalian codon availability andto remove viral elements shown to compromise expression.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flowchart showing a Phase 1 clinical study design whereinall vaccinations with the non-optimized HTNV DNA vaccine and theoptimized PUUV DNA vaccine were administered intradermally using the ND10 PMED device;

FIG. 2A is a graph of neutralizing antibody titers measured in serumsamples collected from subjects vaccinated with the non-optimized HTNVvaccine;

FIG. 2B is a graph of neutralizing antibody titers measured in serumsamples collected from subjects vaccinated with the optimized PUUVvaccine;

FIG. 3 is a graph showing neutralizing antibodies in blood samplescollected on multiple days from two subjects vaccinated with either theoptimized PUUV vaccine (subject 0026) or the non-optimized HTNV vaccine(subject 0029) with each symbol representing the neutralizing antibody(PRNT₅₀) titer for subjects listed in the legend and arrows indicatingvaccination days, The graph shows that the optimized PUUV vaccineinduced neutralizing antibodies to PUUV (closed squares) but not to HTNV(open squares). Likewise, the non-optimized HTNV vaccine inducedantibodies to HTNV (open circles), but did not cause a rise inantibodies to PUUV (closed circles) even though the subject had lowpre-existing antibodies to PUUV (dashed line).

FIG. 4A is a graph showing neutralizing antibody responses to HTNV ofsubjects receiving separate administrations of the non-optimized HTNVDNA vaccine and the optimized PUUV vaccine with each symbol representingthe neutralizing antibody (PRNT₅₀) titer for subjects listed in thelegend and arrows indicating vaccination days, showing that separateadministrations of both the HTNV and PUUV DNA vaccines to a singleindividual can induce antibody responses to HTNV;

FIG. 4B is a graph showing neutralizing antibody responses to PUUV ofsubjects receiving separate administrations of the non-optimized HTNVDNA vaccine and the optimized PUUV vaccine with each symbol representingthe PRNT₅₀ titer for subjects listed in the legend and arrows indicatingvaccination days, showing that separate administrations of both the HTNVand PUUV DNA vaccines to a single individual can induce antibodyresponses to PUUV;

FIG. 5 is a graph showing two individuals vaccinated with the optimizedPUUV DNA that developed antibodies to both HTNV and PUUV;

FIG. 6 is a plasmid map of the synthetic gene HTN-M_CO, consisting of3415 base pairs assembled from synthetic oligonucleotides and/or PCRproducts into a pWRG7077 plasmid;

FIG. 7A is a digital photograph showing HTNV protein (green color)produced by the optimized HTNV M segment vaccine (HTNV M) and detectedby immunofluorescent antibody staining with a monoclonal antibody toHTNV;

FIG. 7B is a digital photograph showing HTNV protein (green color)produced by the non-optimized HTNV M segment vaccine (HTNV M (x) anddetected by immunofluorescent antibody staining with a monoclonalantibody to HTNV;

FIG. 7C is a graph from a flow cytometry assay showing expression of theoptimized HTNV M segment DNA synthesized to also contain the extraneousnucleotides found in the non-optimized HTNV M vaccine as compared to thesame DNA synthesized without the extraneous sequences;

FIG. 7D is a graph from a flow cytometry assay showing expression of thesynthetic optimized HTNV M segment DNA compared to the non-optimizedDNA;

FIG. 8A is a graph showing neutralizing antibodies to PUUV of hamstersvaccinated with the optimized PUUV DNA vaccine or a 1:1 mixture of theoptimized PUUV vaccine and the non-optimized HTNV using two types ofIM-EP devices or one type of intradermal (ID) EP device, showing thatHTNV DNA does not interfere with PUUV DNA immunogenicity;

FIG. 8B is a graph showing neutralizing antibodies to HTNV of hamstersvaccinated with the non-optimized HTNV DNA vaccine alone, the optimizedPUUV DNA vaccine alone or 1:1, 2:1 or 10:1 mixtures of the non-optimizedHTNV DNA and the optimized PUUV DNA vaccines using two types ofintramuscular IM-EP devices or one type of ID-EP device, showing thatPUUV DNA does interfere with HTNV DNA immunogenicity;

FIG. 9 is a graph showing immunogenicity (specific neutralizingantibodies) in hamsters of optimized HTNV DNA vaccine of the inventioncompared to the non-optimized HTNV DNA, the optimized PUUV DNA vaccineor a 1:1 mixture of both optimized DNA vaccines delivered to hamsters byID-EP, showing that the optimized HTNV DNA has overcome interferenceproblems associated with the non-optimized HTNV DNA;

FIG. 10 is a graph showing the immunogenicity (neutralizing antibodiesto HTNV) in hamsters vaccinated by electroporation with the optimizedHTNV DNA vaccine alone, a 1:1 mixture of the optimized HTNV and PUUV DNAvaccines, or the optimized PUUV DNA vaccine alone after each of threesequential vaccinations, indicating that the optimized HTNV vaccine andthe mixture of the optimized HTNV and PUUV DNA vaccines induceneutralizing antibodies to HTNV, but that the optimized PUUV DNA vaccinealone does not elicit neutralizing antibodies to HTNV.

FIG. 11A is a graph showing neutralizing antibodies to PUUV ofindividual rabbits vaccinated by intramuscular electroporation withnon-optimized HTNV DNA vaccine (pWRG/HTN-M(x) as compared to the PUUVDNA vaccine (pWRG/PUU-M(s2) or a mixture of the two vaccines(Combination) showing equivalent neutralizing antibody titers to PUUV;

FIG. 11B is a graph showing neutralizing antibodies to HTNV ofindividual rabbits vaccinated by IM-EP with non-optimized HTNV DNAvaccine (pWRG/HTN-M(x) as compared to the PUUV DNA vaccine(pWRG/PUU-M(s2) or a mixture of the two vaccines (Combination) showingevidence of interference;

FIG. 12A is a graph showing neutralizing antibody responses to HTNV ofhumans vaccinated with the non-optimized HTNV DNA vaccine by IM-EP;

FIG. 12B is a graph showing neutralizing antibody responses to PUUV ofhumans vaccinated with the optimized PUUV DNA vaccine by IM-EP;

FIG. 12C is a graph showing neutralizing antibody responses to HTNV ofhumans vaccinated with a 1:1 mixture of non-optimized HTNV and optimizedPUUV DNA vaccines by intramuscular electroporationIM-EP; indicatingreduced number of responses to HTNV and evidence of interference withHTNV immunogenicity.

FIG. 12D is a graph showing neutralizing antibody responses to PUUV ofhumans vaccinated with a 1:1 mixture of non-optimized HTNV and optimizedPUUV DNA vaccines by intramuscular electroporationIM-EP; indicating nointerference with PUUV immunogenicity.

FIG. 13 is a graph showing a hamster study showing the results ofgeometric mean titers of neutralizing antibodies to each of fourhantaviruses known to cause hemorrhagic fever with renal syndrome inhamsters vaccinated with the optimized HTNV DNA vaccine alone, theoptimized PUUV DNA vaccine alone or a mixture of the optimized HTNV andPUUV DNA vaccines indicating that the optimized HTNV vaccine inducesantibodies to HTNV, SEOV, and DOBV, but not to PUUV); the optimized PUUVvaccine elicits antibodies only to PUUV, but that the mixture of theoptimized HTNV and PUUV vaccines induces neutralizing antibodies to allfour of the hantaviruses;

FIG. 14 is a table showing a study design for an ongoing clinical studyin 120 subjects vaccinated with two doses and at two schedules by IM-EPwith the mixed optimized HTNV and PUUV DNA vaccines;

FIG. 15A is a graph showing showing pseudovirion neutralization titersfrom the clinical study in individual 001 responding to both of theoptimized vaccines delivered as a mixture by IM-EPe;

FIG. 15B is a graph showing showing pseudovirion neutralization titersin individual 013 responding to both vaccines;

FIG. 15C is a graph showing showing pseudovirion neutralization titersin individual 015 responding to both vaccines;

FIG. 15D is a graph showing showing pseudovirion neutralization titersin individual 022 responding to both vaccines;

FIG. 15E is a graph showing showing pseudovirion neutralization titersin individual 023 responding to both vaccines;

FIG. 15F is a graph showing showing pseudovirion neutralization titersin individual 024 responding to both vaccines;

FIG. 16 is a graph showing neutralizing antibody responses in 26 vaccinerecipients four weeks after the third vaccination with the mixedoptimized HTNV and PUUV DNA vaccine by IM-EP, indicating high antibodyresponses to both viruses in some individuals; and

FIG. 17 is a Timeline graph of the Phase 2a study.

DETAILED DESCRIPTION

A recombinant DNA-based vaccine for HTNV and PUUV M segments constructscircumvents key issues associated with both production and formulationof combination vaccines for HFRS. The invention provides a bivalentvaccine for all HFRS-causing viruses, which includes both HTNV and PUUVM segment constructs.

The invention is a new synthetic, codon-optimized HTNV full-length Mgene open reading frame (ORF) that encodes amino acids forming viralproteins. The optimization of the gene has solved a long felt need inthis type of vaccine, namely major gene related interference with formervaccines, which prevented development of a comprehensive vaccine forHFRS. Determining how to optimize and produce a synthetic gene for theHTNV M segment required extensive testing.

This synthetic gene was cloned into a plasmid to form the first HTNVfull-length M gene that elicits neutralizing antibodies in animals whendelivered in combination with a similarly optimized PUUV DNA vaccine(U.S. patent publication US2010/0323024A1, incorporated herein byreference). In addition, the invention obviates the need for anextraneous gene sequence that was previously found to be required forexpression of the non-optimized HTNV gene. The synthetic gene isengineered into a molecular vaccine system to prevent HFRS caused byinfection with HTNV, SEOV or DOBV. Alternatively, it can be combinedwith the optimized PUUV DNA vaccine to protect against HFRS caused byany hantavirus.

Specifically, the invention consists of a genetically modified DNAvaccine representing the open reading frame of the M genome segment ofthe HTNV that has been optimized to include several features known toincrease mammalian expression. See SEQ ID NO. 1

The HTNV DNA vaccine expresses the envelope protein genes of HTNV thatwere adapted to the codon bias of Homo sapiens genes. The codon adaptionindex, which describes how well the codons match the codon usagepreference of homo sapiens, where 1.0 is perfect, was increased for theHTNV gene from 0.67 to 0.97. In addition, regions of the very high(>80%) or very low (<30%) guanine-cytosine (GC) content were avoided inthe genes where possible as either extreme results in poor expression.For the HTNV gene, the average GC content was increased from 40% to 60%,to prolong mRNA half-life. Also, negative cis-acting motifs, such assplice sites, poly(A) signals, TATA-boxes, etc. which may negativelyinfluence expression were eliminated where possible. The optimized HTNVgene open reading frame was then synthesized by Geneart, Inc.(Regensburg, Germany) and inserted between the NotI and BglIIrestriction sites of plasmid backbone pWRG7077 [30] (to create the DNAvaccine construct that comprise the invention (See FIG. 6). The pWRG7077plasmid backbone (pWRG7077 (4326 bp) (PowderJect Vaccines, Inc.,Madison, Wis.) contains the human cytomegalovirus immediate early (CMVIE) promoter with its associated Intron A, a bovine growth hormonetranscription terminator and polyadenylation signal (BGH pA), a pUC19origin of replication (ori), and a kanamycin resistance marker (KanR)shown in FIG. 6. The complete nucleotide sequence of the final HTNV DNAvaccine construct has been confirmed as shown in SEQ ID No. 1.

The optimized HTNV DNA vaccine produces HTNV protein that can berecognized in immunofluorescent antibody assays and by flow cytometrywhen reacted with a monoclonal antibody to a HTNV envelope glycoprotein(FIG. 7A). In addition, the HTNV gene that comprises the invention showscomparable expression in cell cultures to the previously developed,non-optimized HTNV M DNA vaccine, which has non-coding extraneousnucleotides that are required for expression (FIG. 7B). The optimizedgene in this invention does not require the extraneous nucleotides forexpression, and addition of these nucleotides to the construct does notimprove expression. As shown in FIG. 7C, a flow cytometry comparison ofthe optimized HTNV M that were synthesized with or without theextraneous nucleotides had the same expression profiles. Therefore, theinvention is an improvement over the earlier DNA vaccine construct inthat it does not require the presence of noncoding nucleotides toproduce HTNV proteins.

In FIG. 7D, flow cytometry was performed in order to compare expressionof the non-optimized (original) HTNV M DNA to the optimized (HTNV_M_CO)DNA. Two curves are shown, which represent independent analysis. Thesynthetic gene has a similar expression profile as that of the originalgene. Conclusions were that the synthetic gene produces HTNV proteinsthat are recognized by antibodies to authentic HTNV and expression doesnot require the presence of the extraneous nucleotides that must bepresent for expression of the original gene.

Of primary importance for this invention, the new synthetic DNA vaccineconstruct solves a major gene-related interference problem, whichprevented development of a comprehensive vaccine for HFRS. That is, inorder to elicit protective immunity against all four hantaviruses thatare able to cause HFRS, it is necessary to vaccinate with both the HTNVDNA vaccine and also with the PUUV DNA vaccine [28, 29]. However, whenthe native M segment HTNV DNA vaccine was delivered to test animals incombination with the PUUV M segment DNA vaccine, the animals developedantibody responses only to the PUUV component (FIG. 8, and [31]). It wasnot possible to overcome this interference by increasing the ratio ofHTNV to PUUV DNA in the mixture (FIG. 8B).

In FIGS. 8A and 8B, there is shown immunogenicity of HTNV DNA vaccinesdelivered to hamsters using two types of IM-EP devices or one type ofID-EP device. In FIG. 8A, shown are geometric mean titers (GMT) ofneutralizing antibodies to PUUV measured by plaque reductionneutralization test (PRNT) of serum from hamsters vaccinated withoptimized PUUV DNA or with a 1:1 mixture of non-optimized HTNV andoptimized PUUV DNA. The hamsters develop neutralizing antibodies to PUUVin all groups, indicating that non-optimized HTNV DNA does not interferewith optimized PUUV DNA immunogenicity. In FIG. 8B, there is shown GMTof neutralizing antibodies to HTNV measured by PRNT of serum fromhamsters vaccinated with non-optimized HTNV DNA or with 1:1, 2:1 or 10:1mixtures of non-optimized HTNV and optimized PUUV DNA or with optimizedPUUV DNA. Results show that the non-optimized HTNV vaccine elicitsneutralizing antibodies to HTNV in hamsters, but that the optimized PUUVvaccine and the mixed vaccines do not, indicate in the PUUV vaccinealone cannot elicit antibody responses to HTNV in hamsters and thatmixing the PUUV vaccine with the non-optimized HTNV vaccine results ininterference with the immunogenicity of the non-optimized HTNV vaccine.Titers shown are the reciprocal of the dilution of sera required toreduce plaque counts of controls by 50%.

In contrast, using this invention, it is possible to obtain neutralizingantibodies against both HTNV and PUUV in animals that receive the mixedvaccine. In addition, this new optimized HTNV DNA vaccine is at least aseffective or more effective than the non-optimized HTNV DNA vaccine ateliciting antibody responses against HTNV when given alone. See FIG. 9.More specifically, in FIG. 9, there is shown the immunogenicity ofoptimized HTNV of the invention and PUUV DNA vaccines (US 2010/0323024A1), incorporated herein in its entirety by reference, delivered tohamsters by ID-EP. Geometric mean titers (GMT) of neutralizingantibodies to homologous viruses (first three bars) were measured usingsera from hamsters vaccinated with the codon-optimized HTNV DNA vaccine,the non-optimized HTNV DNA vaccine, or the codon-optimized PUUV DNAvaccine. GMT titers to HTNV (fourth bar) or to PUUV (last bar) weremeasured in sera from hamsters vaccinated with a 1:1 mixture of theoptimized HTNV and PUUV M segment optimized DNA vaccines. Titers shownare the reciprocal of the dilution of sera required to reduce plaquecounts of controls by 50%.

Delivery:

To accelerate the immune response to the vaccines, the vaccine isdelivered using a state-of-the art technology component, electroporation(EP). The DNA is formulated in an excipient approved for human delivery,such as sterile normal saline or other inert substance as a carrier.Both intramuscular (IM) and intradermal (ID) EP devices are availableand both have been found to notably enhance the immunogenicity of theHFRS vaccines in animals. ID-EP delivery may be used, which not onlycapitalizes on the efficient delivery of EP, but also offers theadvantages of reduced cost and logistics for mass vaccinations. Thisbivalent vaccine, in combination with EP delivery accelerates the immuneresponse to the hantaviruses and reduce the number of dosings needed toachieve protective immunity as compared to delivery without EP.

Other delivery methods include jet injection and nanoparticleencapsulation.

To measure the safety of the vaccine in controlled studies under GoodLaboratory Practice (GLP) conditions, rabbits are vaccinated with eitherIM-EP or ID-EP of the optimized vaccine given alone or in combinationwith the PUUV DNA vaccine. Two manufacturers' EP devices have beentested with the hantavirus DNA vaccines (Ichor and Inovio) in hamstersand both have produced excellent results. IM-EP has been tested moreextensively in humans than ID-EP, and is currently the gold standarddelivery method for DNA vaccines; however, ID-EP has been found toelicit stronger immune responses than IM-EP for some pathogens becauseskin is a highly immunologically active organ with numerous circulatingantigen presenting cells. In addition to possibly improvingimmunogenicity with ID-EP, skin vaccination is a desired deliveryplatform for mass vaccination with biodefense vaccines. Current clinicalIM-EP delivery requires loading of DNA vaccine into the delivery deviceat the time of delivery, whereas the ID-EP platform consists ofpreloaded disposable cartridges containing the DNA vaccines, which canbe administered using a re-useable EP device. The prototype ID-EP devicehas already been tested in a successfully completed GLP non-clinicalsafety study in rabbits and humans with another biodefense-related DNAvaccine for Venezuelan equine encephalitis virus.

Together the vaccine and EP delivery platform proposed offers expedientscale-up, long term stability, reduced cold-chain requirements, and massvaccination applicability.

Safety Study:

A safety study in rabbits was used to obtain approval for testing of thecombined non-optimized HTNV and optimized PUUV DNA vaccines in humans. Ahuman study was also recently completed with no serious adverse eventsrelated to the vaccines reported. Similarly, a second safety study, alsoto be performed in rabbits, will be used in support of a pending IM-EPvs ID-EP Phase 1 clinical study with the optimized HTNV DNA vaccinealone and in combination with the optimized PUUV vaccine. The rabbitstudy characterizes local and/or systemic adverse responses associatedwith optimized HTNV and/or PUUV vaccine candidates administered usingthe IM-EP and ID-EP devices. A summary of the repeat dose safety andtoxicity study design is shown in Table 1.

TABLE 1 Summary of Repeat Dose Safety/Toxicity Study Design Injection #& Admin. N Group Vaccine Delivery Dose Volume Schedule (M/F) Endpoint 1HTNV IM-EP 3.0 mg 1 × 1000 μl 0, 14, 28, 56 20 5 M/5 F: Day 58 (10/10) 5M/5 F: Day 70 2 HTNV ID-EP 1.2 mg 2 × 200 μl  0, 14, 28, 56 20 5 M/5 F:Day 58 (10/10) 5 M/5 F: Day 70 3 HTNV + IM-EP 6.0 mg 1 × 1000 μl 0, 14,28, 56 20 5 M/5 F: Day 58 PUUV (10/10) 5 M/5 F: Day 70 4 HTNV + ID-EP2.4 mg 2 × 200 μl  0, 14, 28, 56 20 5 M/5 F: Day 58 PUUV (10/10) 5 M/5F: Day 70 5 PUUV ID-EP 1.2 mg 2 × 200 μl  0, 14, 28, 56 20 5 M/5 F: Day58 (10/10) 5 M/5 F: Day 70 6 Vehicle IM-EP Saline 1 × 1000 μl 0, 14, 28,56 20 5 M/5 F: Day 58 (carrier) & (IM) (10/10) 5 M/5 F: Day 70 ID-EP 2 ×200 μl  (no EP) (ID)

The invention uses cGMP manufacturing for both the safety study inrabbits and the pending clinical study. The cGMP manufacturing isconducted at a contract research organization and includes extensiverelease testing for potency, purity and stability, prior to use in thePhase 1 clinical trial.

Clinical Trial

To assess the safety of the optimized HTNV and PUUV DNA vaccines, 6groups of 10 subjects each for a total of 60 subjects and 12 alternatesubjects are vaccinated with the optimized HTNV vaccine, the optimizedPUUV vaccine or a mixture of both vaccines. Subjects in one groupreceive the HTNV DNA vaccine candidate administered using the TDS-IM-EPdelivery device (3.0 mg dose). Two other groups receive either the HTNVor PUUV DNA vaccines delivered by the ID-EP device (0.6 mg dose), andtwo groups receive the HTNV-PUUV mixed vaccine candidate administeredusing the IM-EP (6.0 mg total dose) or ID-EP device (1.2 mg total dose).Ten subjects receive a placebo control (5 by ID-EP, 5 by IM-EP). Notethat differences in dose levels for the two routes of administration aredue to the difference in volume of injection that will be administeredby the respective routes (0.2 ml ID versus 1.0 ml IM).

Example 1: (Non-Optimized HTNV Study, in Which HTNV and PUUV Vaccinesare Delivered as Separate Administrations)

Candidate DNA vaccines for hemorrhagic fever with renal syndromeexpressing the envelope glycoprotein genes of Hantaan (HTNV) or Puumala(PUUV) viruses were evaluated in an open-label, single-center Phase 1study consisting of three vaccination groups of nine volunteers. Thevolunteers were vaccinated by particle-mediated epidermal delivery(PMED) three times at four-week intervals with the HTNV DNA vaccine, thePUUV DNA vaccine or both vaccines. At each dosing, the volunteersreceived 8 μg DNA/4 mg gold. There were no study-related serious adverseevents, and all injection site pain was graded as mild. The mostcommonly reported systemic adverse events were fatigue, headache,malaise, myalgia, and lymphadenopathy. Blood samples were collected ondays 0, 28, 56, 84, 140, and 180, and assayed for the presence ofneutralizing antibodies. In the single vaccine groups, neutralizingantibodies to HTNV or PUUV were detected in 30% or 44% of individuals,respectively. In the combined vaccine group, 56% of the volunteersdeveloped neutralizing antibodies to one or both viruses. These resultsdemonstrate that the HTNV and PUUV DNA vaccines are safe and can beimmunogenic in humans when delivered as separate administrations by PMED(FIGS. 1, 2A, 2B, 3, 4A, 4B, 5).

As shown in FIG. 3, neutralizing antibodies to PUUV detected in allsamples of two subjects, indicating that there was a pre-existingexposure to a hantavirus prior to the start of this study; But when theperson with the pre-existing antibodies to PUUV was vaccinated with thenon-optimized HTNV vaccine, they had minimal response to thenon-optimized HTNV vaccine, providing evidence that there is no boost inantibody response (i.e. interference in developing HTNV antibodies) evenin individuals that have pre-existing antibodies to PUUV;

Example 2: (Non-Optimized HTNV Study, in Which the HTNV and PUUV DNAVaccines are Given as a Mixture, Resulting in Interference)

In this study, vaccines were delivered using Ichor medical System'sIM-EP device. The study included 3 randomized groups of 9 subjects, eachof whom received three vaccinations at days 0, 28, and 56 with 2 mg ofDNA/1 mL of the non-optimized HTNV vaccine, the optimized PUUV vaccine,or a mixture of both vaccines. Three vaccinations were given four weeksapart. No serious adverse events related to the vaccine were observed.Analysis of blinded serum samples indicated that neutralizing antibodieswere elicited against both HTNV and PUUV, but that in volunteersreceiving both vaccines, interference was observed, with only threesubjects developing neutralizing antibodies to HTNV (FIG. 12C) ascompared to seven developing neutralizing antibodies to PUUV (FIG. 12D).

Example 3: (Non-Optimized HTNV DNA Preclinical Safety Study in RabbitsShowing that Mixed HTNV and PUUV DNA Vaccines Result in Reduced Responseto HTNV in Rabbits, i.e., Interference)

In FIGS. 11A and 11B, results from a GLP safety study of non-optimizedHTNV DNA vaccine and optimized PUUV DNA are shown. The vaccine deliveredto rabbits by electroporation was performed. Rabbits were vaccinatedthree times by IM-EP with individual or mixed vaccines (1 mg each) andneutralizing antibodies were measured. Control rabbits were vaccinatedwith phosphate buffered saline (PBS) using IM-EP for delivery. The datashow that equivalent neutralizing antibody titers to PUUV were elicitedwhen the PUUV vaccine, pWRG/PUUM(s2), is given alone or mixed with thenon-optimized HTNV DNA vaccine, pWRG/HTNM(x) (FIG. 11A); however,greatly reduced titers to HTNV were observed with the mixed vaccines ascompared to those obtained with the HTNV DNA vaccine alone (FIG. 1B).

Example 4: (Optimized HTNV Study in Hamsters Showing that Interferencehas Been Overcome Using the Inventive Optimized HTNV DNA Vaccine)

FIG. 9 and FIG. 10 demonstrate the immunogenicity of the inventiveoptimized HTNV DNA vaccine when mixed 1:1 with PUUV DNA vaccine (US2010/0323024A1) delivered to hamsters by IM-EP in two separate studies.The hamsters were vaccinated three times at 3-4 week intervals by IM-EPwith 100 μg of the individual DNAs or with a 1:1 mixture the optimizedHTNV and PUUV DNAs (50 μg of each). Geometric mean antibody titers(GMT₅₀) for each group of eight hamsters are shown as the reciprocal ofthe dilution of sera required to reduce plaque counts of controls by50%. As expected, because half as much of the optimized HTNV DNA wasgiven in the mixture as compared to in those receiving only theoptimized HTNV DNA vaccine, the antibody response to HTNV was slightlyreduced. Following injection of HTNV into the vaccinated hamsters, itwas determined that none of the hamsters vaccinated with the mixedvaccines showed evidence of infection with HTNV (as determined bymeasuring antibodies to the N protein, which is not part of the vaccine,data not shown). Consequently, the mixed vaccines containing theinventive optimized HTNV DNA vaccine can elicit neutralizing antibodiesagainst HTNV and also protect from infection by HTNV. Moreover, therewas no detectable interference observed when the inventive HTNV DNAvaccine was mixed with the PUUV DNA vaccine.

Example 5: (Non-Optimized HTNV DNA Vaccine Phase 1 Clinical Study UsingIM-EP Delivery, Showing that Mixed HTNV and PUUV DNA Vaccines Result inReduced Response to HTNV, i.e., Interference)

FIG. 12 A-D show neutralizing antibody responses of volunteersvaccinated by IM-EP with the individual or mixed non-optimized HTNV andoptimized-PUUV DNA vaccines. Seven of eleven volunteers that werevaccinated with the non-optimized HTNV DNA vaccine developedneutralizing antibodies to HTNV. Six of eight volunteers vaccinated withoptimized PUUV vaccine developed neutralizing antibodies to PUUV. Threeof nine volunteers vaccinated with the combination vaccine developedantibodies to both HTNV and PUUV. However four additional volunteers hadantibodies only to PUUV but no additional volunteers had antibodies onlyto HTNV. Interference, while not complete, is still a problem. Theseresults are in line with the results in Example 2.

Preparation of Optimized DNA Vaccines

The optimized HTNV DNA vaccine was constructed by cloning cDNArepresenting the optimized HTNV M segment open reading frame, whichencodes Gn and Gc, into the NotI and BglII-restriction sites of pWRG7077[14] as described previously [8]. The PUUV DNA vaccine was previouslyconstructed similarly, using cDNA that was engineered as a consensussequence of several PUUV strains, and codon-optimized (GeneArt) [15] and(US 2010/0323024A1). The HTNV and PUUV DNA vaccines were produced undercurrent Good Manufacturing Practices (cGMP) by Althea Technologies, Inc.(San Diego, Calif.). A summary of the manufacturing and testingprocesses that Althea was contracted to perform is as follows:

Optimized HTNV DNA vaccine plasmid is manufactured under cGMPspecification, to include (a) Establishment and Characterization of aManufacturer's Master Cell Bank (MCB) (b) Process Optimization & Non-GMPProduction of a HTNV plasmid DNA vaccine lot, (c) cGMP Production andCharacterization of a bulk HTNV Plasmid DNA (2.6 g) (d) Packaging andShipment of Cell Banks (2 SHIPMENTS), (e) 6 months Bulk Drug ProductStorage. Deliverables requested from Althea are (1) a Master cell bankfor HTNV, (2) a pilot lot of HTNV DNA plasmid; (3) one cGMP lot of bulkDNA plasmid stored in IPA (2.6 g); and (4) all documents (e.g., batchrecords, data records and reports, CoAs, BMF letter of cross reference)as required for submission to FDA.

The following specific tasks are performed by Althea for manufacture ofa bulk DNA vaccine plasmid for HTNV.

Example 6: (Optimized HTNV Vaccine)

As shown in FIG. 13, gene modifications to overcome interference weremade. As previously performed for the PUUV plasmid, the codons of theHTNV plasmid were modified to the bias of homo sapiens genes, and knownmotifs that reduce expression were removed. Hamsters vaccinated with theoptimized vaccine of the invention by ID-EP three times at 3-weekintervals developed neutralizing antibodies to all four HFRS-causinghantaviruses.

Example 7

FIG. 14 shows a study design for 120 people that receive a mixedoptimized vaccine. Two different doses are given (2 mg vs 1 mg) and 2different schedules with each individual receiving an optional 6-monthboost.

FIGS. 15a-15f show examples of pseudovirion neutralization titers invarious individuals from the study of FIG. 14 responding to bothoptimized HTNV and PUUV vaccines. Vesicular stomatitis viruses werepseudo typed with optimized HTNV or PUUV glycoproteins and used toassess neutralizing antibody responses of Phase 2a vaccine recipients atindicated days post vaccination. The individuals of FIGS. 15a-15f arealso represented in FIG. 16.

Example 8

FIG. 16 also shows results from the study design of FIG. 14. FIG. 16shows neutralizing antibody responses detected in sera of the first 26vaccine recipients four weeks after the third vaccination with optimizedHTNV and PUUV DNA vaccines by IM-EP. As samples are blinded untilcompletion of this study, it is not possible to assess dose or routefactors with these samples. The neutralizing antibody titers weremeasured using a traditional plaque reduction neutralization test (PRNT)or a vesicular stomatitis virus pseudo typed with the glycoproteins ofoptimized HTNV or PUUV (PsVNA). The bars show each person's neutralizingantibodies to optimized Hantaan or Puumala viruses as measured by thetwo different tests. The first test uses authentic HTNV (plaquereduction neutralization test) and the second test uses a nonpathogenicvirus (VSV) that is coated with HTN proteins (pseudovirionneutralization assay). This second assay can be completed quickly and atBSL2 rather than BSL3 conditions.

Summary:

Phase 1 study results show that DNA vaccines expressing the envelopeglycoprotein genes of HTNV and PUUV are safe and immunogenic in humanswhen delived by IM-EP.

Animal studies suggest that immune interference between the HTNV andPUUV plasmids can be resolved using gene-optimized plasmids. Dose andschedule studies are in progress using the optimized plasmids.

Preliminary (blinded) analysis of sera from a subset of Phase 2a timepoints (FIG. 17) shows that in the first 26 vaccinees, the overallseroconversion rate is 92% with 81% positive for optimized HTNV and 88%positive for PUUV at one or more time points.

Establishment of Master Cell Bank (MCB)

Includes:

Preparation of MCB batch record

Transformation

Selection, genetic stability

Growth

Dispensing/freezing (no less than 200 vials)

Characterization of MCB

Includes:

Completion of MCB testing outlined in Attachment 1

QA review of associated testing

Generation of C of A

Non-GMP Production/Process Optimization

Includes:

Evaluation of plasmid in Althea's fermentation and purificationprocesses

Non-GMP plasmid will be provided to USAMRMC for research use only

Appearance, size, and identity of plasmid by restriction analysis usingtwo common enzymes

Purity by gel electrophoresis

A260/280 (1.7/2.0)

Endotoxin by LAL

An optimization study must be completed prior to GMP Production

cGMP Production and Characterization of Plasmid DNA (2.6 g)

Includes:

Establishment of specifications

Preparation of customized cGMP Manufacturing Batch Records

Optimization of fermentation conditions

Fermentation

Development of large scale purification process

Cell lysis

Downstream processing and separation

Column purification

Preparation of standard bulk

In-process testing

Shipping

Packaging and Shipment of Cell Banks (2SHIPMENTS)

Includes the management and preparation of the cell banks

Includes verification of shipment products from Althea to a USAMRMCspecified destination

Includes temperature controlled shipping containers with temperatureloggers

Preparation of all required shipping documentation

Shipping on Dry Ice

Bulk Drug Product Storage (6 Months)

Includes temperature monitored storage of Bulk Drug Product.

pWRG7077HTN-M-CO DNA of M segment and plasmid together SEQ ID NO. 1GGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCGGCATGCCTGCAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCaATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACACCCCTTT GGCTCTTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACACCCCCGCTTCCTTATGCTATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCCCTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTATCTCTATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTACAGGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGTCCCCCGTGCCCGCAGTTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCCACATCCGAGCCCTGGTCCCATGCCTCCAGCGGCTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCACAGCACAATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCTCGGAGATTGGGCTCGCACCGCTGACGCAGATGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTATTCTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCGTCCAAGCTTGCGGCCGCCACCATGGGCATCTGGAAGTGGCTGGTCATGGCTAGCCTCGTGTGGCCCGTGCTGACCCTGCGGAACGTGTACGACATGAAGATCGAGTGCCCCCACACCGTGTCCTTCGGCGAGAACAGCGTGATCGGCTACGTGGAACTGCCCCCCGTGCCCCTGGCCGATACAGCTCAGATGGTGCCCGAGAGCAGCTGCAGCATGGACAACCACCAGAGCCTGAACACCATCACCAAGTACACCCAGGTGTCCTGGCGGGGCAAGGCCGATCAGAGCCAGAGCAGCCAGAACAGCTTCGAGACAGTGTCTACCGAGGTGGACCTGAAGGGCACCTGTGCCCTGAAGCACAAGATGGTGGAAGAGAGCTACCGGTCCAGAAAGAGCGTGACCTGCTACGACCTGAGCTGCAACAGCACCTACTGCAAGCCCACCCTGTACATGATCGTGCCCATCCACGCCTGCAACATGATGAAGTCCTGCCTGATCGCCCTGGGCCCCTACAGAGTGCAGGTCGTGTACGAGCGGAGCTACTGCATGACCGGCGTGCTGATCGAGGGCAAGTGCTTCGTGCCCGACCAGAGCGTGGTGTCCATCATCAAGCACGGCATCTTCGATATCGCCAGCGTGCACATCGTGTGCTTTTTCGTGGCCGTCAAGGGCAACACCTACAAGATTTTCGAGCAGGTCAAAAAGAGCTTCGAGAGCACCTGTAACGACACCGAGAACAAGGTGCAGGGCTACTACATCTGCATCGTGGGCGGCAACAGCGCCCCCATCTACGTGCCCACCCTGGACGACTTCCGGTCCATGGAAGCCTTCACCGGCATCTTCAGAAGCCCCCACGGCGAGGACCACGACCTGGCCGGCGAGGAAATCGCCAGCTACTCCATCGTGGGCCCTGCCAACGCCAAGGTGCCACACAGCGCCAGCAGCGACACCCTGTCCCTGATCGCCTACAGCGGCATCCCCAGCTACAGCAGCCTGAGCATCCTGACCAGCAGCACCGAGGCCAAGCACGTGTTCAGCCCTGGCCTGTTCCCCAAGCTGAACCACACCAACTGCGACAAGAGCGCCATCCCCCTGATCTGGACCGGCATGATCGACCTGCCCGGCTACTACGAGGCCGTGCACCCCTGCACCGTGTTCTGCGTGCTGTCTGGCCCTGGAGCCAGCTGCGAGGCCTTTTCTGAGGGCGGCATCTTTAACATCACCAGCCCCATGTGCCTGGTGTCCAAGCAGAACCGGTTCCGGCTGACCGAGCAGCAGGTCAACTTCGTGTGCCAGCGGGTGGACATGGACATCGTGGTGTACTGCAACGGCCAGCGGAAAGTGATCCTGACCAAGACCCTCGTGATCGGCCAGTGCATCTACACCATCACAAGCCTGTTCAGCCTGCTGCCCGGCGTGGCCCACTCTATCGCCGTGGAACTGTGCGTGCCCGGCTTTCACGGCTGGGCCACAGCTGCCCTGCTGGTCACCTTCTGCTTCGGCTGGGTGCTGATCCCCGCCATCACCTTCATCATCCTGACCGTGCTGAAGTTTATCGCCAACATCTTCCACACCAGCAACCAGGAAAACCGGCTCAAGTCCGTGCTGCGGAAGATCAAAGAGGAATTCGAAAAGACCAAGGGCAGCATGGTCTGCGACGTGTGCAAATACGAGTGCGAGACATACAAAGAGCTGAAGGCCCACGGCGTGTCCTGCCCTCAGAGCCAGTGCCCCTACTGCTTCACCCACTGCGAGCCTACCGAGGCCGCCTTCCAGGCCCACTACAAAGTGTGCCAGGTCACACACCGGTTCAGGGACGACCTGAAGAAAACCGTGACCCCCCAGAACTTCACCCCCGGCTGCTACCGGACCCTGAACCTGTTCCGGTACAAGAGCCGGTGCTACATCTTTACCATGTGGATCTTTCTGCTGGTGCTCGAGTCCATCCTGTGGGCCGCCAGCGCCAGCGAAACCCCTCTGACCCCCGTGTGGAACGACAACGCCCATGGCGTGGGCTCTGTGCCCATGCACACCGACCTGGAACTGGACTTCAGCCTGACCAGCTCCAGCAAGTACACCTACCGGCGGAAGCTGACCAACCCCCTGGAAGAGGCCCAGAGCATCGACCTGCACATCGAGATCGAGGAACAGACCATCGGAGTCGATGTCCACGCCCTGGGACATTGGTTCGACGGACGGCTGAACCTGAAAACCAGCTTCCACTGCTACGGCGCCTGCACTAAGTACGAGTACCCCTGGCACACCGCCAAGTGCCACTACGAGCGGGACTACCAGTACGAGACAAGCTGGGGCTGTAACCCCAGCGACTGTCCAGGCGTGGGCACCGGCTGTACAGCTTGTGGCCTGTACCTGGACCAGCTGAAGCCCGTGGGCTCCGCCTACAAGATCATCACCATCCGGTACAGCAGACGCGTGTGCGTGCAGTTCGGCGAAGAGAACCTGTGCAAGATCATCGACATGAACGACTGCTTCGTGTCCCGGCACGTGAAAGTGTGCATCATCGGCACCGTGTCCAAGTTCAGCCAGGGCGATACCCTGCTGTTCTTCGGCCCTCTGGAAGGCGGCGGACTGATCTTCAAGCACTGGTGCACAAGCACCTGTCAGTTTGGCGACCCCGGCGACATCATGAGCCCCAGAGACAAGGGCTTCCTGTGCCCCGAGTTCCCCGGCAGCTTCCGGAAGAAGTGCAACTTCGCCACCACCCCCATCTGCGAGTACGACGGCAACATGGTGTCCGGCTACAAGAAAGTGATGGCCACCATCGACAGCTTCCAGAGCTTCAACACCTCCACCATGCACTTCACCGACGAGCGGATCGAGTGGAAGGACCCCGACGGCATGCTGCGGGACCACATCAACATCCTGGTCACCAAGGACATCGACTTCGACAACCTGGGCGAGAACCCCTGCAAGATCGGCCTGCAGACCTCCAGCATCGAGGGCGCTTGGGGCAGCGGCGTGGGCTTTACCCTGACCTGTCTGGTGTCCCTGACCGAGTGCCCCACCTTCCTGACCTCCATCAAGGCCTGCGACAAGGCCATCTGTTACGGCGCCGAGTCCGTGACCCTGACAAGAGGCCAGAACACCGTGAAGGTGTCCGGCAAAGGCGGCCACAGCGGCAGCACCTTCAGATGCTGCCACGGGGAGGACTGCAGCCAGATCGGACTGCATGCCGCAGCACCCCACCTGGACAAAGTGAACGGCATCAGCGAGATCGAGAACTCCAAGGTGTACGACGATGGCGCCCCTCAGTGCGGCATCAAGTGTTGGTTCGTGAAGTCCGGCGAGTGGATCAGCGGCATCTTCTCCGGCAACTGGATCGTGCTGATTGTGCTGTGCGTGTTCCTGCTGTTTAGCCTGGTGCTGCTGAGCATTCTGTGTCCCGTGCGCAAGCACAAGAAAAGCTGATGAAGATCTACGTATGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGACAGCTCGACTCTAGaATTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC HTN-M-CO Optimized DNA of M segmentSEQ ID NO. 2 ATGGGCATCTGGAAGTGGCTGGTCATGGCTAGCCTCGTGTGGCCCGTGCTGACCCTGCGGAACGTGTACGACATGAAGATCGAGTGCCCCCACACCGTGTCCTTCGGCGAGAACAGCGTGATCGGCTACGTGGAACTGCCCCCCGTGCCCCTGGCCGATACAGCTCAGATGGTGCCCGAGAGCAGCTGCAGCATGGACAACCACCAGAGCCTGAACACCATCACCAAGTACACCCAGGTGTCCTGGCGGGGCAAGGCCGATCAGAGCCAGAGCAGCCAGAACAGCTTCGAGACAGTGTCTACCGAGGTGGACCTGAAGGGCACCTGTGCCCTGAAGCACAAGATGGTGGAAGAGAGCTACCGGTCCAGAAAGAGCGTGACCTGCTACGACCTGAGCTGCAACAGCACCTACTGCAAGCCCACCCTGTACATGATCGTGCCCATCCACGCCTGCAACATGATGAAGTCCTGCCTGATCGCCCTGGGCCCCTACAGAGTGCAGGTCGTGTACGAGCGGAGCTACTGCATGACCGGCGTGCTGATCGAGGGCAAGTGCTTCGTGCCCGACCAGAGCGTGGTGTCCATCATCAAGCACGGCATCTTCGATATCGCCAGCGTGCACATCGTGTGCTTTTTCGTGGCCGTCAAGGGCAACACCTACAAGATTTTCGAGCAGGTCAAAAAGAGCTTCGAGAGCACCTGTAACGACACCGAGAACAAGGTGCAGGGCTACTACATCTGCATCGTGGGCGGCAACAGCGCCCCCATCTACGTGCCCACCCTGGACGACTTCCGGTCCATGGAAGCCTTCACCGGCATCTTCAGAAGCCCCCACGGCGAGGACCACGACCTGGCCGGCGAGGAAATCGCCAGCTACTCCATCGTGGGCCCTGCCAACGCCAAGGTGCCACACAGCGCCAGCAGCGACACCCTGTCCCTGATCGCCTACAGCGGCATCCCCAGCTACAGCAGCCTGAGCATCCTGACCAGCAGCACCGAGGCCAAGCACGTGTTCAGCCCTGGCCTGTTCCCCAAGCTGAACCACACCAACTGCGACAAGAGCGCCATCCCCCTGATCTGGACCGGCATGATCGACCTGCCCGGCTACTACGAGGCCGTGCACCCCTGCACCGTGTTCTGCGTGCTGTCTGGCCCTGGAGCCAGCTGCGAGGCCTTTTCTGAGGGCGGCATCTTTAACATCACCAGCCCCATGTGCCTGGTGTCCAAGCAGAACCGGTTCCGGCTGACCGAGCAGCAGGTCAACTTCGTGTGCCAGCGGGTGGACATGGACATCGTGGTGTACTGCAACGGCCAGCGGAAAGTGATCCTGACCAAGACCCTCGTGATCGGCCAGTGCATCTACACCATCACAAGCCTGTTCAGCCTGCTGCCCGGCGTGGCCCACTCTATCGCCGTGGAACTGTGCGTGCCCGGCTTTCACGGCTGGGCCACAGCTGCCCTGCTGGTCACCTTCTGCTTCGGCTGGGTGCTGATCCCCGCCATCACCTTCATCATCCTGACCGTGCTGAAGTTTATCGCCAACATCTTCCACACCAGCAACCAGGAAAACCGGCTCAAGTCCGTGCTGCGGAAGATCAAAGAGGAATTCGAAAAGACCAAGGGCAGCATGGTCTGCGACGTGTGCAAATACGAGTGCGAGACATACAAAGAGCTGAAGGCCCACGGCGTGTCCTGCCCTCAGAGCCAGTGCCCCTACTGCTTCACCCACTGCGAGCCTACCGAGGCCGCCTTCCAGGCCCACTACAAAGTGTGCCAGGTCACACACCGGTTCAGGGACGACCTGAAGAAAACCGTGACCCCCCAGAACTTCACCCCCGGCTGCTACCGGACCCTGAACCTGTTCCGGTACAAGAGCCGGTGCTACATCTTTACCATGTGGATCTTTCTGCTGGTGCTCGAGTCCATCCTGTGGGCCGCCAGCGCCAGCGAAACCCCTCTGACCCCCGTGTGGAACGACAACGCCCATGGCGTGGGCTCTGTGCCCATGCACACCGACCTGGAACTGGACTTCAGCCTGACCAGCTCCAGCAAGTACACCTACCGGCGGAAGCTGACCAACCCCCTGGAAGAGGCCCAGAGCATCGACCTGCACATCGAGATCGAGGAACAGACCATCGGAGTCGATGTCCACGCCCTGGGACATTGGTTCGACGGACGGCTGAACCTGAAAACCAGCTTCCACTGCTACGGCGCCTGCACTAAGTACGAGTACCCCTGGCACACCGCCAAGTGCCACTACGAGCGGGACTACCAGTACGAGACAAGCTGGGGCTGTAACCCCAGCGACTGTCCAGGCGTGGGCACCGGCTGTACAGCTTGTGGCCTGTACCTGGACCAGCTGAAGCCCGTGGGCTCCGCCTACAAGATCATCACCATCCGGTACAGCAGACGCGTGTGCGTGCAGTTCGGCGAAGAGAACCTGTGCAAGATCATCGACATGAACGACTGCTTCGTGTCCCGGCACGTGAAAGTGTGCATCATCGGCACCGTGTCCAAGTTCAGCCAGGGCGATACCCTGCTGTTCTTCGGCCCTCTGGAAGGCGGCGGACTGATCTTCAAGCACTGGTGCACAAGCACCTGTCAGTTTGGCGACCCCGGCGACATCATGAGCCCCAGAGACAAGGGCTTCCTGTGCCCCGAGTTCCCCGGCAGCTTCCGGAAGAAGTGCAACTTCGCCACCACCCCCATCTGCGAGTACGACGGCAACATGGTGTCCGGCTACAAGAAAGTGATGGCCACCATCGACAGCTTCCAGAGCTTCAACACCTCCACCATGCACTTCACCGACGAGCGGATCGAGTGGAAGGACCCCGACGGCATGCTGCGGGACCACATCAACATCCTGGTCACCAAGGACATCGACTTCGACAACCTGGGCGAGAACCCCTGCAAGATCGGCCTGCAGACCTCCAGCATCGAGGGCGCTTGGGGCAGCGGCGTGGGCTTTACCCTGACCTGTCTGGTGTCCCTGACCGAGTGCCCCACCTTCCTGACCTCCATCAAGGCCTGCGACAAGGCCATCTGTTACGGCGCCGAGTCCGTGACCCTGACAAGAGGCCAGAACACCGTGAAGGTGTCCGGCAAAGGCGGCCACAGCGGCAGCACCTTCAGATGCTGCCACGGGGAGGACTGCAGCCAGATCGGACTGCATGCCGCAGCACCCCACCTGGACAAAGTGAACGGCATCAGCGAGATCGAGAACTCCAAGGTGTACGACGATGGCGCCCCTCAGTGCGGCATCAAGTGTTGGTTCGTGAAGTCCGGCGAGTGGATCAGCGGCATCTTCTCCGGCAACTGGATCGTGCTGATTGTGCTGTGCGTGTTCCTGCTGTTTAGCCTGGTGCTGCTGAGCATTCTGTGTCCCGTGCGCAAGCACAAGA AAAGCTGATGA pWRG7077plasmid SEQ ID NO. 3 GGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCGGCATGCCTGCAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCaATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACACCCCTTT GGCTCTTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACACCCCCGCTTCCTTATGCTATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCCCTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTATCTCTATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTACAGGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGTCCCCCGTGCCCGCAGTTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCCACATCCGAGCCCTGGTCCCATGCCTCCAGCGGCTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCACAGCACAATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCTCGGAGATTGGGCTCGCACCGCTGACGCAGATGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTATTCTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCGTCCAAGCTTGCGGCCGCCACCAGATCTACGTATGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGACAGCTCGACTCTAGaATTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC SEQ ID NO. 4 Bgl II cloning site AGATCTSEQ ID NO. 5 Not I cloning site GCGGCCGC

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What is claimed is:
 1. A bivalent vaccine for HTNV and PUUV thatprotects against HFRS caused by HTNV infection comprising a syntheticcodon-optimized HTNV full-length M gene open reading frame segment ofSEQ ID NO. 1 and PUUV DNA vaccine.
 2. The bivalent vaccine of claim 1,wherein immunogenic amounts of said open reading frame of SEQ ID No. 1and said PUUV DNA are administered using intramuscular or intradermaldelivery devices.
 3. The bivalent vaccine for HTNV and PUUV of claim 1,wherein said PUUV DNA is optimized in dose and delivery schedule.